調控啤酒酵母四異戊二烯焦磷酸合成酶雙元體裂解成單元體的可能機制以及人類四異戊二烯焦磷酸合成酶形成六元體的研究

Abstract

四異戊二烯焦磷酸合成酶 (geranylgeranyl pyrophosphate synthase) 可催化一個異戊二烯焦磷酸(isopentenyl pyrophosphate)與法呢基焦磷酸 (farnesyl pyrophosphate) 反應產生四異戊二烯焦磷酸 (geranylgeranyl pyrophosphate)，此產物是四異戊二烯化 (geranylgeranylated) 蛋白質、胡蘿蔔素 (carotenoid)、細胞膜脂質等－這些在生物體內重要分子的前趨物。不同於其他已知的異戊二烯轉移酶 (trans-prenyltransferase) ，啤酒酵母的四異戊二烯焦磷酸合成酶具有一延伸至另一單元體 (subunit) 的N端胺基酸鍊。在先前的研究中，我們已經了解第八號白胺酸與第九號異白胺酸對於雙元體的形成有不可或缺的貢獻 (羅嘉翔，未發表實驗結果)。第八號白胺酸及第九號異白胺酸突變為甘胺酸的雙定點突變造成啤酒酵母的四異戊二烯焦磷酸合成酶雙元體裂解成單元體，並且使得活性下降約103倍 (羅嘉翔，未發表實驗結果)。為了進一步研究第八號白胺酸及第九號異白胺酸突變為甘胺酸造成啤酒酵母的四異戊二烯焦磷酸合成酶雙元體裂解成單元體的可能機制，我觀察此蛋白質的立體結構，提出了二個可能機制，藉由與第八號白胺酸及第九號異白胺酸形成網絡連結來穩定雙元體的結構 (圖四及圖五)。其中一個機制為:第八號白胺酸及第九號異白胺酸與第一百六十三號白胺酸及第一百六十七號甲硫胺酸間具有凡得瓦爾作用力，藉此固定相鄰第一百六十八號天冬醯胺酸、第一百六十九號離胺酸及第一百七十一號甘胺酸的位置，而這三個胺基酸又與位在雙元體間主要介面的第一百四十一號甘胺酸、第一百三十七號天冬醯胺酸及第一百三十八號白胺酸間有凡得瓦爾作用力，藉此穩固介面的結構 (圖四)。尤其，第一百六十九號離胺酸與第一百四十五號天冬胺酸間有淨電荷作用力，而第一百四十五號天冬胺酸又能與第一百四十一號甘胺酸利用蛋白質骨架形成氫鍵，第一百四十一號甘胺酸又與第一百三十七號天冬醯胺酸形成氫鍵，而這兩者都位在雙元體間主要介面處。此穩定的雙元體界面使得靠近第一百三十八號白胺酸與第一百四十一號甘胺酸的第一百三十九號組胺酸得以與另一單元體中的第一百零一號天冬醯胺酸形成氫鍵 (圖四)。另一個可能的機制為:第八號白胺酸及第九號異白胺酸與第兩百號白胺酸及第兩百零三號異白胺酸間具有凡得瓦爾力，藉此固定第一百九十九號天冬醯胺酸的位置，此胺基酸與第一百七十五號精胺酸間形成氫鍵，而第一百七十五號精胺酸與位在雙元體間主要介面的第一百三十四號麩胺酸間具有淨電吸引力，藉以固定雙元體介面的結構 (圖五)。由於第一百七十五號精胺酸與第一百三十四號麩胺酸都與第一百七十九號精胺酸形成氫鍵，所以更強化了上述的作用力 (圖五)。我以單定點突變法將上述第一個可能機制中的第一百六十三號白胺酸、第一百六十七號甲硫胺酸、第一百零一號天冬醯胺酸轉變為甘胺酸，第一百四十五號天冬胺酸轉變為離胺酸，並不能將雙元體瓦解為單元體。同樣以單定點突變法將第二個可能機制中的第兩百號白胺酸與第兩百零三號異白胺酸轉變為甘胺酸，第一百三十四號麩胺酸與第一百七十五號精胺酸轉變為丙胺酸，也無法將雙元體瓦解為單元體。然而，以雙定點突變法將牽涉到第一個可能機制的第一百六十七號甲硫胺酸轉變為甘胺酸，同時將牽涉到第二個可能機制的第一百九十九號天冬醯胺酸轉變為丙胺酸，所得到的突變蛋白同時含有單元體與雙元體。這顯示上述所提出的二個機制同時調控酒酵母四異戊二烯焦磷酸合成酶由雙元體裂解成單元體。 人類的四異戊二烯焦磷酸合成酶不具有一延伸至另一單元體的N端胺基酸鍊，然而卻能形成穩定的六元體。觀察此蛋白質的立體結構，我們發現N端的第一個

Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipids. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. In previous study, we have known that L8 and I9 were essential in dimerization (Lo, C. H. et al., unpublished data). The double mutant L8G/I9G can completely disrupt a dimeric S. cerevisiae GGPPs into a monomer with at least 103-fold reduction in activity (Lo, C. H. et al., unpublished data). In order to investigate mechanisms by which L8G/I9G disrupt the dimer, I observed the X-ray crystal structure of the enzyme and proposed that two sets of interactions networking with L8 and I9 may stabilize the dimerization (Figure 4 and Figure 5). One mechanism is that L8 and I9 make vdW contacts with L163 and M167, whose neighboring residues, N168, K169, and G171, are within vdW contact of G141, N137 and L138 located at the main dimer interface (Figure 4). In particular, K169 forms a salt-bridge with D145, which in turn forms a backbone-backbone hydrogen bond with G141, which itself is hydrogen bonded to N137, both of which are at the main dimer interface (Figure 4). H139, close to L138 and G141, forms a hydrogen bond with N101 in the other chain (Figure 4). The other is that L8 and I9 make vdW contacts with L200 and I203 whose neighboring residue, N199, is hydrogen bonded to R175, which in turn forms a salt-bridge with E134 located near the main dimer interface (Figure 5). This interaction is reinforced since R175 and E134 are also both hydrogen bonds to R179 (Figure 5). I then performed site-directed mutagenesis studies and used analytic ultracentrifuge (AUC) to characterize the mutants. Single mutant of L163G, M167G, D145K, or N101G involved in one possible mechanism and L200G, I203G, E134A, or R175A involved in the other did not result in the disruption of dimer to monomer, whereas double mutant of M167G/N199A involved in both possible mechanisms resulted in a mixture of dimer and monomer. This indicated that both possible mechanisms were involved in modulating disruption of S. cerevisiae geranylgeranyl pyrophosphate synthase dimer into monomer. In human geranylgeranyl pyrophosphate synthase, there is no N-terminal arm protruding into the other subunit, whereas it forms a stable hexamer. The 3-D structure of human GGPPs suggested that the first helix at N-terminus may play an important role in inter-dimer interaction to stabilize the structure of hexamer. To confirm this, the human GGPPS without the first 21 amino acids were characterized using analytic ultracentrifugation (AUC).This resulted in a mixture of monomer and dimer. To identify the critical amino acids in the N-terminal helix for hexamerization, I observed the X-ray crystal structure of the enzyme and assumed E14, Y18, or Q21 may involved in inter-dimer interactions by binding with Y246 and T228 to stabilize the hexamer. To confirm this, I generated E14G/Y18G/Q21G, E14G, Y18G, and Q21G and characterized them respectively using AUC. The triple mutant E14G/Y18G/Q21G became a mixture of monomer and tetramer, whereas the single mutant E14G, Y18G, and Q21G remained as a hexamer. The results suggested that E14, Y18, and Q21 together provided critical roles for the hGGPPs inter-dimer interactions and stabilize the structure of hexamer.